The goal of this proposal is to understand how two receptor tyrosine kinases (RTKs) with Wnt-binding domains in their extracellular regions mediate and/or contribute to Wnt signaling. Wnts are secreted ligands of approximately 330 amino acids that play key roles in embryonic development and control a spectrum of processes in the adult, ranging from organ regeneration to stem cell maintenance to neural circuit generation to sex determination. They do this by regulating many cellular processes including cell proliferation, migration, polarity, apoptosis, differentiation and survival. Not unexpectedly, therefore, aberrations in Wnt signaling are now known to be involved in many diseases, including bone diseases, cancers, diabetes, neurodevelopment disorders, and several congenital malformations. In addition to the best known Wnt receptors (the Frizzleds), two additional Wnt receptor families have been identified: the Ryk family (Ryk in humans, Derailed, Derailed-2 and Doughnut in Drosophila) and the Ror family (Ror1 and Ror2 in human, DRor and DNrk in Drosophila). Both are receptor tyrosine kinase (RTK) families - a receptor class not typically associated with Wnt signaling, but a class that has been successfully targeted therapeutically in other signaling systems with kinase inhibitors and antibody therapeutics. The extracellular regions of Ryk and Ror family RTKs contain domains typically associated with binding to Wnt ligands. Ryks contain a Wnt Inhibitory Factor-1 (WIF) domain, and Rors contain an extracellular cysteine-rich domain (CRD) that resembles the Wnt-binding CRDs in the Frizzled receptors. We propose here to investigate the signaling mechanisms of Ryk and Ror family RTKs using an array of approaches from in vivo assays in Xenopus embryos and cellular studies to biochemical, biophysical and structural approaches. Our goal is to determine whether Ryk and Ror receptors resemble other well known RTKs in their mode of signaling, or instead function as co-receptors for Wnts with Frizzleds or other molecules. In our Specific Aims, we will address the following questions: 1. how do Ryk family members recognize their Wnt ligands, and do Ryks resemble other RTKs in their response to extracellular ligand binding? 2i. Do Ror family members resemble other RTKs in responses to ligand binding, and does Wnt binding by their cysteine-rich domains (CRDs) resemble that of Frizzleds? 2ii. Do Ryks and Rors heterodimerize, and can Wnts bind simultaneously to both WIF domains and CRDs to drive co-receptor heterodimerization? These studies will provide important fundamental new insight into how these novel classes of Wnt receptors signal, which is crucial for understanding Wnt signaling specificity and teasing out its multiple roles. In addition, our findings should open potential new avenues for therapeutic inhibition - as the roles of these Wnt-binding RTKs in disease become increasingly clear.